Flow cytometry support to research in immune, skin, muscle and bone diseases

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

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Abstract

Summary This flow cytometry core facility supports the research of the majority of NIAMS IRP investigators. In addition, it is actively involved in cytometer training, data analysis and flow cytometric assay development for users new to this technique. The NFCS currently houses and maintains the following instrumentation for the use of NIAMS investigators: -BD Biosciences FACSAria Fusion cell sorter (5-laser excitation, 19-color detection, Automated Cell Deposition Unit, Biosafety Cabinet) -BD Biosciences FACSAria IIIu cell sorter (5-laser excitation, 19-color detection, Automated Cell Deposition Unit, Aerosol Management Option) -BD Biosciences Influx cell sorter (7-laser excitation, 20-color detection, automated cell deposition unit, spectral analyzer, Biosafety Cabinet) -BD Biosciences FACSCanto SORP analyzer with HTS (4-laser excitation, 13-color detection, high-throughput sampler) -BD Biosciences LSR Fortessa SORP analyzer with HTS (7-laser excitation, 20-color detection, high-throughput sampler) -BD Biosciences FACSVerse analyzer with UL (3-laser excitation, 8-color detection, universal loader for multi-well plates or tubes, volumetric measurement for absolute cell counts) -BD Biosciences FACSymphony A5 analyzer (5-laser, 28-color detection, HTS-ready, advanced electronics) -BD FACSCelesta SORP analyzer with HTS (4-laser, 15-color detection, high-throughput sampler, advanced electronics) The following instruments are no longer housed and maintained by NFCS but are still accessible to NIAMS investigators: -Compucyte iCys laser scanning cytometer (3-laser excitation, 5-parameter detection), shared with NCI -Amnis ImageStreamX Mark II high resolution imaging cytometer (7-laser excitation, 3 imaging objectives, IdeasTM analysis software) In years past the FCS core has housed and utilized other cytometers (listed below) to support the NIAMS mission. These have since been retired and replaced with the more modern instruments listed above. -Beckman Coulter MoFlo high-speed cell sorter -Beckman Coulter CyAn ADP analyzer -BD Biosciences FACSCalibur anylzer -BD Biosciences FACS Vantage DiVa high-speed cell sorter -BD Biosciences LSR I analyzer In addition to the projects from those laboratories focused on mechanisms and dysfunctions of the immune system, the facility continues to provide flow cytometry and sorting services to NIAMS laboratories engaged in disciplines not typically served by flow cytometry such as bone, muscle and skin biology. The facility also has the capability of sorting patient specimens and human-derived cells with a higher degree of safety. Following the addition of the FACSAria Fusion cell sorter, housed in a laminar flow biosafety cabinet, the existing BD InFlux cell sorter likewise gained its own biosafety cabinet during the core facility relocation in June 2014. These cell sorters now provide more biosafe opportunities for NIAMS investigators to further the Bench to Bedside initiative promoted by the NIH Director, Dr. Francis Collins. To provide the NFCS with even more capable tools, the 5-laser, 28-color BD FACSymphony A5 and 4-laser, 15-color BD FACSCelesta SORP analyzers were added to the core facility in 2018. These state-of-the-art cytometers with their more sensitive detection electronics enable investigators to take advantage of the synergy of higher dimensional immunophenotyping and potentially discover previously undetectable rare cell populations. Although the instruments have been moved to other institutes, investigators whose research involves specimens that are not well suited to flow cytometry can still take advantage of a laser scanning cytometer, the Thorlabs (Compucyte) iCys, as well as the Amnis ImageStreamX Mark II imaging flow cytometer. These microscope-based instruments provide fluorescent and morphological data on cells, tissue fixed to slides, multi-well plates (iCys), or cells in suspension as in traditional flow cytometry (ImageStreamX). Along with immunophenotyping, apoptosis assays and co-localization studies, the iCys can also re-analyze specific cells over time for kinetics-based assays. In contrast, the design of the ImageStreamX Mark II allows for higher throughput for more robust statistics, a characteristic of standard flow cytometers, while providing the image analysis advantage of a microscope-based system. The FCS has three dedicated personnel to support the needs of NIAMS IRP investigators: -Mr. Jeffrey Lay has been an integral part of the NFCS since 2008. He is technically trained in the use of all analyzers and cell sorters and is the primary operator of the BD InFlux. He actively and routinely handles complete core operation, equipment inventories, actively promotes the utilities of core operation, guidance and learning, and actively immerse himself with new technologies. -Kevin Tinsley, PhD Having completed postdoctoral fellowship in the field of immunology at NCI, he joined the NFCS with substantial experience with flow cytometry as a contractor in 2013. He subsequently completed his FACSAria Operator training at BD Biosciences, was promoted to Staff Scientist and is the primary operator of the FACSAria IIIu.Additionally, he regularly takes courses through FAES in the area of Data Science to increase his understanding of much of the data analysis done in NIAMS. -Mr. James Simone, Section Leader, took over the management of the NFCS in 2003 and continues to oversee the day-to-day operations of the facility. He is expert in the use of all the facility flow cytometers and continues to be involved in the promotion of flow cytometry at the NIH and within the Mid-Atlantic Region. As a co-chair of the NIH Flow Cytometry Interest Group, he assists in organizing and promoting quarterly meetings that showcase leading researchers in the field and promote relationships with vendors of flow cytometry-related products. His longtime membership in the International Society for the Advancement of Cytometry (ISAC) and the ISAC Biosafety Committee helps facilitate the exchange of new ideas and information with peers from around the world. More than 350 NIAMS flow cytometry users have been trained to date on the various cytometers available in the facility. All new users attend a general introduction and orientation to facility equipment and policies. Then, according to needs and previous experience, they progress to individualized or group instruction on the instruments most appropriate for their particular research needs. After approval by the section leader, users can then operate the instruments unassisted. In all cases, however, the NFCS staff is available for technical support and consultation. To further support the NIAMS mission, the NIAMS Flow Cytometry Section continues to investigate new techniques and applications in cytometry for the use of its investigators including sorting for single-cell PCR, DNA and RNA expression and single-cell Western (Milo, Protein Simple). Finally, due to the new NIH CREx (Collaborative Research Exchange) advocated by Drs. Collins and Gottesman, we can now make cytometry services available to investigators outside of NIAMS.